Polarizing light microscopes have been used for approximately 200 years to study, among other things, characteristics of crystalline materials and other ordered materials. More complex instruments, referred to as “circular dichroism spectrapolarimeters,” are used to measure circular dichroism (“CD”) in optically active materials, including proteins and other biological materials. There are four different phenomena observed when plain polarized or circularly polarized light is passed through optically active materials: (1) linear birefringence (“LB”), a phase shift between propagation modes of linearly polarized light resulting from anisotropic refraction of light by an optically anisotropic material; (2) linear dichroism (“LD”), resulting from anisotropic absorption of linearly polarized light passing through an optically active sample; (3) circular birefringence (“CB”, also known as optical activity or optical rotation), resulting from a difference in refractive index of a sample with respect to left circularly polarized light and right circularly polarized light; and (4) circular dichroism (“CD”), resulting from differential absorption of left circularly polarized light and right circularly polarized light by an optically active sample. Often, two or more of these phenomena are convolved in light propagating through various media, producing complex observed effects that were formerly difficult to analyze. During the past 50 years, relatively straightforward mathematical descriptions of these polarization-related phenomena have been developed and have allowed for development of instruments and computational methods for detecting, deconvolving, and quantifying LB, LD, CB, and CD signals in a variety of instruments in which polarized light is passed through samples. As one example, Metripol® produces a polarizing-light-microscope system for detecting LB.
Detection and quantification of LB, LD, CB, and CD signals can provide useful information in a wide variety of different applications. For example, CD signals generated from protein samples are related to the presence of optically active secondary and tertiary structure within the protein samples, and provide a means for characterizing dynamic conformational changes within a protein sample. Polarization effects in biological samples may be used for image-contrast purposes as well as for detecting dynamically changing macromolecular structures and polymer orientations related to a wide variety of different biological effects and phenomena. In one recently recognized application, real-time detection of LB signals in sample wells in which crystals are grown provides the basis for automated crystal detection and may facilitate massive crystallization efforts needed for high-volume and high-throughput molecular structure determination by x-ray crystallography that is a cornerstone of current efforts in proteomics, structural genomics, and structural biology.
Unfortunately, current polarizing microscopy techniques rely on relatively complex hardware involving mechanical rotation of samples and/or polarizers as well as on relatively intensive computational analysis of multiple captured images in order to produce a final image that indicates the presence or absence of an LB signal at discrete locations within the image. These methods are currently too slow, cumbersome, and expensive for use in automated detection of crystals, real-time biological-sample imaging, and many other uses. Researchers, developers, and equipment vendors have thus recognized the need for a real-time imaging system that reveals and quantifies LB signals within images collected by polarizing microscopy and other techniques.